The present invention is directed to methods for screening for HTLV-I and HTLV-II.
Human T cell lymphotropic virus type I (HTLV-I) is known to be the cause of neoplastic diseases, such as adult T cell leukemia/lymphoma (ATLL), as well as of fatal demyelinating disorders, such as tropical spastic paraparesis and HTLV-I-associated myelopathy (TSP/HAM) (Gessain et al, 1985; Osame et al, 1987), which are non-neoplastic inflammatory/autoimmune conditions. While the majority of affected patients have antibodies, more recent studies have identified HTLV-I antibody negative cases of ATLL (Poiesz et al, 1980; Hinuma et al, 1981; Miyoshi et al, 1981) and TSP/HAM cases occur (Korber et al, 1991; Ohshima et al, 1991; Ramirez et al, 1998). Transmission of the virus is mediated by transfusion of virus-harboring leukocytes in blood and breast milk, as well as sexually, from male to female. Perinatal transmission occurs also in 5% of non-breast-fed children of healthy virus-carrying mothers (Gessain et al, 1985). In contrast to HIV, free virus particles do not seem to be infectious. However, following transfusion of cellular blood components harboring HTLV-I, 44 to 64% of recipients seroconvert (Osame et al, 1986; Blattner, 1989; Yoshida, 1994. For this reason, all blood used for transfusion in the USA has been screened for antibodies to the structural proteins of HTLV since 1988. This is done using ELISA or Western blot techniques using viral lysates which identify antibodies to the structural proteins of the virus, such as HTLV-I/II core (gag) protein p24 and envelope (Env) proteins gp46, gp64/68.
On the basis of the above testing, the prevalence of HTLV-I infection among Americans without obvious risk factors, such as being from an endemic region or intravenous drug abuse, has been estimated to be about 0.016% (Lee et al, 1991). It may be as high as 0.1% in some locations (Williams et al, 1988). One suspects that infection with HTLV-I infection among Caucasian Americans may be higher than determined by routinely used methods in light of studies on cutaneous T cell lymphoma mycosis fungoides (MF) (Zucker-Franklin et al, 1991; Pancake et al, 1995). MF patients usually do not have antibodies to the structural proteins of the virus, but harbor proviral sequences of HTLV-I in their peripheral blood mononuclear cells and skin (Zucker-Franklin, 1991; Pancake et al, 1995; Zucker-Franklin, 1994; Khan et al, 1996). A high percentage of these patients were also shown to have antibodies to HTLV-I Tax (Pancake et al, 1996b), an antigen not included in commercially available HTLV serologic tests.
A study was conducted of healthy relatives of MF patients who were serologically negative for antibodies to HTLV-I when their specimens were tested at a major blood transfusion center (Zucker-Franklin et al, 1997a). Of the first eight individuals tested, six proved to have tax sequences in the PBMCs and antibodies to the Tax antigen by Western blot analysis. Since family studies of a relatively rare disease manifested mostly in middle-aged and elderly individuals are time-consuming, a more expeditious approach was chosen to determine whether currently used serologic methods are adequate to establish the true prevalence of HTLV infection among blood donors.
Accordingly, a cohort of individuals among whom the prevalence of HTLV infection was known to be high, i.e., known injection drug users (IDUs) was selected for study (Zucker-Franklin et al, 1997b). Matched sera and PBMCs obtained from 81 HIV-negative methadone clinic attendees were tested by routine serologic methods, as well as for tax, pol, and gag proviral sequences by PCR/Southern blot analysis and antibodies to viral structural proteins, as well as to the Tax gene product. Routine serology proved 18/81 (22%) of these specimens to be positive for antibodies to HTLV, which concurred with results obtained by other investigators. On the other hand, 39/81 (48.1%) were found positive for HTLV proviral sequences by biomolecular means, and 42 (51.8%) were positive when both serologic tests and PCR/Southern blot analyses were used. Together, the results of these studies suggested that the prevalence of infection with HTLV, particularly when efforts are made to detect Tax sequences, may be considerably higher than is currently believed.
Food and Drug Administration-approved blood screening assays are available which may be used to detect the presence of HTLV-I antibodies in blood samples. Available screening assays are discussed in CDCP (1988). These assays typically use viral antigenic proteins isolated from mammalian cell cultures which are infected with HTLV-I. Other assays are reported in Sawada et al, U.S. Pat. No. 4,588,681; Essex et al, PCT Publication WO 84/04327; Copeland et al (1986); Saxinger et al (1983); Bodner et al, EPA 1035352; and Hare et al, PCT Publication WO 91/07510, the entire contents of all of which are hereby incorporated by reference.
However, many people infected with HTLV-I or -II have lost the sequences which encode the structural components of the virus. While these individuals have antibodies to Tax, they test negative for antibodies to HTLV by tests which are currently used in blood banks (Zucker-Franklin et al, 1997a; Zucker-Franklin et al, 1997b; Ehrlich, 1989; Shiori, 1993; Pancake, 1996b). Thus, their blood is considered safe for transfusion.
Some of the problems associated with use of HTLV-I proteins derived from infected mammalian cells may be overcome by applying recombinant DNA methods and techniques to develop antigenic polypeptides in non-mammalian host cells.
HTLV-I assays using recombinant antigenic polypeptides have been described. Lal et al (1996) have made antigenic polypeptides. Antigenic polypeptides expressed in E. coli transformed with portions of the gag gene may be used in an immunodot assay. The sensitivity of this immunodot assay was described as being comparable to Western blots, and the results were described as being as reliable as radioimmunoassays (Kanner et al, 1986). Itoh et al, in U.S. Pat. No. 4,795,805, disclose another assay using antigenic polypeptides encoded by the gag gene.
WO 91/07510 of Amgen Inc. discloses the use of one or more recombinant polypeptide antigens which are polypeptides encoded by all or part of the env, tax or gag genes of HTLV-I. There is no suggestion therein that the use of any one of these antigens is better than either of the others.
Other HTLV-I derived recombinant antigenic polypeptides have also been used in immunoassays. Cell lysates containing either a 59 kD fusion polypeptide encoded by about half of the env gene and about three-quarters of the tax gene or a single 100 kD fusion polypeptide encoded by gag, env, and tax gene fragments reacted with sera from an HTLV-I infected patient using a Western blot analysis (Kitajima et al, 1988).
A sensitive HTLV-I assay which uses recombinant antigenic polypeptides requires antigens which are readily available and which are immunologically reactive with antibodies found in all or nearly all seropositive individuals. These antigenic polypeptides must be readily purified in order to avoid or eliminate non-specific binding to contaminating host cell proteins by cross-reactive antibodies which may be present in body fluid samples. These antigenic polypeptides must also retain their immunological activity when they are used to prepare immunoassay apparatus which typically involve adsorption of the antigenic polypeptides onto a solid support and contacting the adsorbed polypeptides which various blocking and washing reagents.
It has previously been believed that a sensitive HTLV-I assay requires more than one antigen in a single assay in order to detect individuals exposed to HTLV-I that have different antibody profiles. Hare et al, PCT WO 91/07510, note that for any given seropositive population, individuals exhibit different immunogenic responses to viral antigens, and an assay using only one antigen may not detect all of the exposed individuals. Thus, Hare et al use a single screening assay using more than one antigen in order to ensure all exposed individuals are detected, having found that an immunoassay employing only a single antigen would not be able to accurately identify all infected serum samples (page 9, lines 5-7). This assay is limited to assaying for HTLV-I and requires a separate assay to test for HTLV-II.
Therefore, there is a need for a simple assay which would accurately identify all infected serum samples for HTLV-I and/or HTLV-II infection.
It is an object of the present invention to overcome the aforesaid deficiencies in the prior art.
It is another object of the present invention to provide a single assay for HTLV-I and/or HTLV-II in serum samples.
It is another object of the present invention to provide a method for screening blood donors or potential blood donors for carriers of disease or conditions related to HTLV-I and/or HTLV-II infection.
It is a further object of the present invention to provide a method for screening pregnant women and nursing mothers for infections related to HTLV-I and/or HTLV-II infection.
It is another object of the present invention to provide a method for screening patients for HTLV infection when the patients do not test positive for antibodies to HTLV.
According to the present invention, blood or other body fluid of a given population is screened for infection or past infection with HTLV-I and/or HTLV-II by subjecting each sample to a test for the presence of the HTLV-I or HTLV-II Tax protein, DNA which encodes the Tax protein, or antibodies specific to the subject Tax protein; and correlating the presence of HTLV-I and/or HTLV-II infection with the result of the test. Because this test, which relies on testing for the presence of the HTLV-I or HTLV-II Tax protein, is so specific for HTLV-I and/or HTLV-II infection, there is no requirement for input from any other test result to test positively for HTLV-I and/or HTLV-II.
Use of such a tax test for widespread blood screening has many unexpected advantages over use of the existing blood screening test based on antibodies to structural proteins of the HTLV virus. Not only will the tax test be positive for virtually every individual who tests positively in the standard HTLV test, but one also will discern very important positives which would have tested negative with the conventional HTLV test. Blood which tests tax positive but structural protein negative is particularly harmful as it has been found that recipients of such blood sera convert to tax positive and the tax protein alone may cause health problems. Thus, an important screening test, i.e., that for structural proteins of HTLV, can be eliminated and substituted with the test of the present invention, which will not only test positive when the blood would have tested positive for the standard HTLV test, but will also find another factor which is not found in the standard HTLV test, but for which it is very important to screen out of blood intended for transfusions.
It has now been found that the transforming/transactivating component of the HTLV-I and HTLV-II virus is the tax sequence and its gene product, p40Tax (reviewed in Centers for Disease Control and Prevention, 1988). Currently, the presence of this component is not tested in blood used for transfusion. However, it was reported several years ago that patients with mycosis fungoides, a skin lymphoma, harbor HTLV-I Tax in their circulating and skin-infiltrating lymphocytes (Zucker-Franklin et al, 1991; Pancake et al; 1995; Khan et al, 1996) while being serologically HTLV-I/II negative. These patients, as well as a high proportion of their healthy relatives, have antibodies to the p40Tax protein (Pancake et al, 1995; Pancake et al, 1996b; Zucker-Franklin et al, 1997a). Since one of these healthy relatives admitted to being a frequent blood donor, a study of blood donors who tested HTLV-I/II serologically negative was initiated. It was found that about 8% of such donors harbor HTLV-I tax sequences in their lymphocytes and have antibodies to the p40Tax protein (Zucker-Franklin, 1997b; Zucker-Franklin et al, 1998).
According to recent reports which give methods and further information regarding the prevalence and transmission of Tax alone, it has been found that Tax alone (without the presence of the complete virus) is taken up by, and is able to immortalize cells in vitro, and that it causes lymphomas and fibromas in Tax transgenic mice (Benvenisty et al, 1992; Nerenberge et al, 1987).
The methods which enabled the present inventors to detect Tax in cells of HTLV seronegative individuals and the reasons some investigators failed to identify xe2x80x9cTax onlyxe2x80x9d positive carriers have been published in detail (Nishioka, 1996; Terada et al, 1994; Sumida et al, 1994; Marriette et al, 1993). The present invention provides a method for rapidly and reliably testing for the presence of HTLV-I and/or HTLV-II in specified populations. This is particularly important for screening potential blood donors and pregnant women who are considering nursing their babies.